RESUMEN
N-terminal acetylation in E coli is a rare event catalyzed by three known N-acetyl-transferases (NATs), each having a specific ribosomal protein substrate. Multiple, gram-scale lots of recombinant F1-V, a fusion protein constructed from Y. Pestis antigens, were expressed and purified from a single stably transformed E. coli cell bank. A variant form of F1-V with mass increased by 42-43 Da was detected in all purified lots by electrospray orthogonal acceleration time-of-flight mass spectrometry (MS). Peptide mapping LCMS localized the increased mass to an N-terminal Lys-C peptide, residues 1-24, and defined it as +42.0308+/-0.0231 Da using a LockSpray exact mass feature and a leucine enkaphalin mass standard. Sequencing of the variant 1-24 peptide by LCMS and high-energy collision induced dissociation (LCMS(E)) further localized the modification to the amino terminal tri-peptide ADL and identified the modification as N(alpha)-acetylation. The average content of N(alpha)-acetylated F1-V in five lots was 24.7+/-2.6% indicating that a stable acetylation activity for F1-V was established in the E. coli expression system. Alignment of the F1-V N-terminal sequence with those of other known N(alpha)-acetylated ectopic proteins expressed in E. coli reveals a substrate motif analogous to the eukaryote NatA' acetylation pathway and distinct from endogenous E. coli NAT substrates.
Asunto(s)
Proteínas Bacterianas/análisis , Biotecnología/métodos , Mapeo Peptídico , Proteínas Recombinantes de Fusión/análisis , Espectrometría de Masa por Ionización de Electrospray , Yersinia pestis/química , Acetilación , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Clonación Molecular/métodos , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Yersinia pestis/genéticaRESUMEN
BACKGROUND: Group A streptococcus (GAS) causes illness ranging from uncomplicated pharyngitis to life-threatening necrotizing fasciitis, toxic shock, and rheumatic fever. Attempts to develop an M protein-based vaccine have been hindered by the fact that some M proteins elicit both protective antibodies and antibodies that cross-react with human tissues. New molecular techniques have allowed the previous obstacles to be largely overcome. METHODS: The vaccine is comprised of 4 recombinant proteins adsorbed to aluminum hydroxide that contain N-terminal peptides from streptococcal protective antigen and M proteins of 26 common pharyngitis, invasive, and/or rheumatogenic serotypes. Thirty healthy adult subjects received intramuscular 26-valent GAS vaccine (400 microg) at 0, 1, and 4 months, with clinical and laboratory follow-up for safety and immunogenicity using assays for tissue cross-reactive antibodies, type-specific M antibodies to 27 vaccine antigens, and functional (opsonization) activity of M protein antibodies. RESULTS: The incidence of local reactogenicity was similar to that for other aluminum hydroxide-adsorbed vaccines in adults. No subject developed evidence of rheumatogenicity or nephritogenicity, and no induction of human tissue-reactive antibodies was detected. Overall, 26 of 27 antigenic peptides evoked a >4-fold increase in the geometric mean antibody titer over baseline. The mean log2 fold-increase in serum antibody titer (+/- standard error of the mean) for all 27 antigens was 3.67 +/- 0.21. A significant mean log2 reduction in streptococcal bacterial counts in serum samples obtained after immunization was seen in opsonization assays for all M serotypes. CONCLUSIONS: On the basis of epidemiological data demonstrating that the majority of cases of pharyngitis, necrotizing fasciitis, and other invasive streptococcal infections are caused by a limited number of serotypes, this 26-valent vaccine could have significant impact on the overall burden of streptococcal disease.
Asunto(s)
Infecciones Estreptocócicas/prevención & control , Vacunas Estreptocócicas/inmunología , Adulto , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas Portadoras/inmunología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteínas Recombinantes de Fusión/inmunología , Streptococcus pyogenes/inmunología , Vacunas Sintéticas/inmunologíaRESUMEN
Fluorescent light (FL) photodegradation of a monoclonal antibody (mAb) formulated in histidine buffer is mediated by histidine-derived photosensitizers that accumulate and greatly increase with light exposure. Histidine-derived photosensitizers are the primary mediators of Trp photooxidation. FL-photodegradation requires light exposure and is pH dependent. It is significantly reduced or eliminated by buffer exchanges, by oxygen depletion, or at pH values greater than 7. Antibody-fragment MS ion counts reveal that oxidation of a single light chain Trp in CDR1 correlates with binding loss. Multiple heavy chain methionines oxidize, but poorly correlate with binding loss. Photosensitizers extracted from photo-aged histidine buffer are potent mediators of FL-photodegradation including oxidation and, to a lesser degree, fragmentation and aggregation of the mAb. These photosensitizers absorb visible light and have neutral mass of 187.1- 386.1 Da. They are also fluorescent with ex/em at 360/450 nm. When spiked into histidine or MES buffered mAb formulations they produce a concentration dependent and pronounced increase in FL-photodegradation; however, no oxidation or loss of antibody function occurs in the dark and hydrogen peroxide does not oxidize Trp. The major component is consistent with histidine oxidation to 6a-hydroxy-2-oxo-octahydro-pyrollo[2,3-d]imidazole-5-carboxylic acid. Photosensitizer levels measured in the formulation prior to light exposure, are linearly related to the FL-photodegradation observed and can predict degradation in photostability testing.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Histidina/metabolismo , Inmunoglobulina G/metabolismo , Fotólisis , Fármacos Fotosensibilizantes/metabolismo , Anticuerpos Monoclonales/química , Tampones (Química) , Electroforesis Capilar , Fluorescencia , Histidina/química , Concentración de Iones de Hidrógeno , Inmunoglobulina G/química , Luz , Oxidación-Reducción , Fármacos Fotosensibilizantes/químicaRESUMEN
A modified peptide mapping strategy using electrospray time-of-flight mass spectrometry with high-performance liquid chromatography (HPLC/MS) provides an improved measure of deamidation by performing proteolytic digestion at low temperature (4 degrees C), low pH (6.0) and in organic solvent (> or =10% acetonitrile). HPLC resolution of the native (N) and deamidated (D) peptides is achieved, and the ratio of ion counts is converted into percent deamidation. The percent deamidation is established for a reference lot using a time course of digestion (24-120 h) and extrapolation to time zero. Test samples are compared against the reference lot to quantitate changes in site-specific deamidation. A recombinant purified protein (antigen A) having a single labile Asn-Gly site is analyzed using this strategy. The N and D peptides from an endoproteinase Lys C (Lys C) digestion (pH 6, 4 degrees C) resolve to near homogeneity on HPLC which results in equivalent percent deamidation when calculated by either UV or ion counts. Deamidation increases with time and pH of proteolysis. Lys C peptide maps of antigen A and bovine serum albumin (BSA) digested at pH 5-8 are comparable. A Lys C digestion time course of a reference lot of antigen A extrapolates to 18% deamidation of the Asn-Gly site at time zero. This strategy may be generally applicable to protease-protein combinations for improved accuracy in measuring site-specific deamidation by peptide mapping LC/MS.
Asunto(s)
Algoritmos , Amidas/química , Aminoácidos/química , Cromatografía Líquida de Alta Presión/métodos , Mapeo Peptídico/métodos , Péptidos/química , Espectrometría de Masa por Ionización de Electrospray/métodosRESUMEN
We have characterized protein antigens after quantitative dissociation from aluminum hydroxide adjuvant. Bovine serum albumin (BSA) and a multi-antigen vaccine for Group A Streptococcus (GrAS Vaccine) were formulated on aluminum hydroxide, stored for > or =10 days then eluted with a 48-h treatment at 4 degrees C with 0.85% H(3)PO(4) plus 4M guanidine HCl (GnHCl). BSA is recovered from adjuvant at 92+/-2%. GrAS antigens are equally recovered from GrAS Vaccine (95+/-11% of total protein expected using multiple lots stored for up to 12 months). Recovery after elution is similar when determined by RP-HPLC, SEC-HPLC, UV absorbance, or Bradford methods. Eluted antigens are structurally and functionally intact as judged relative to both treated and untreated antigen controls by SDS-PAGE, RP-HPLC, SEC-HPLC, and after desalting by circular dichroism, bis-ANS binding, and antigenicity determined by ELISA. When formulated and stored for a few weeks, BSA has more dimer (31+/-5%) relative to the elution control (9% dimer) as detected by SEC-HPLC, suggesting that BSA microaggregation is promoted on aluminum. Antigens eluted from very aged GrAS Vaccine (>12 months) show marked changes by RP-HPLC. Structural changes in the antigens under elution conditions were evaluated using bis-ANS, a fluorescent probe of protein structure. Binding of bis-ANS increases fluorescence approximately 100-fold and is significantly diminished with increasing GnHCl concentrations indicating a progressive denaturing of the proteins. At 4M GnHCl (with or without 0.85% H(3)PO(4)) the GrAS antigens are fully denatured and BSA is partially denatured. Interestingly, the addition of 0.85% H(3)PO(4) increases bis-ANS binding on GrAS antigens and reduces the denaturing of GrAS antigens and BSA by chaotropes. Desalting or diluting the eluted antigens results in renaturing of the proteins as judged by bis-ANS fluorescence, circular dichroism and antigenicity testing. The elution method provides a novel approach for high recovery and characterization of GrAS Vaccine antigens and may be applicable to the study of many aluminum hydroxide-bound vaccines.
Asunto(s)
Adyuvantes Inmunológicos/química , Hidróxido de Aluminio/química , Antígenos Bacterianos/química , Antígenos Bacterianos/inmunología , Fenómenos Químicos , Química Farmacéutica , Química Física , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Estabilidad de Medicamentos , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Colorantes Fluorescentes , Desnaturalización Proteica , Proteínas Recombinantes/inmunología , Albúmina Sérica Bovina/química , Espectrofotometría Ultravioleta , Streptococcus pyogenes/inmunologíaRESUMEN
A multivalent vaccine containing amino-terminal M protein fragments from 26 different serotypes of group A streptococci was constructed by recombinant techniques. The vaccine consisted of four different recombinant proteins that were formulated with alum to contain 400 microg of protein per dose. Rabbits were immunized via the intramuscular route at 0, 4, and 16 weeks. Immune sera were assayed for the presence of type-specific antibodies against the individual recombinant M peptides by enzyme-linked immunosorbent assay and for opsonic antibodies by in vitro opsonization tests and indirect bactericidal tests. The 26-valent vaccine was highly immunogenic and elicited fourfold or greater increases in antibody levels against 25 of the 26 serotypes represented in the vaccine. The immune sera were broadly opsonic and were bactericidal against the majority of the 26 different serotypes. Importantly, none of the immune sera cross-reacted with human tissues. Our results indicate that type-specific, protective M protein epitopes can be incorporated into complex, multivalent vaccines designed to elicit broadly protective opsonic antibodies in the absence of tissue-cross-reactive antibodies.
Asunto(s)
Antígenos Bacterianos , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas Portadoras/inmunología , Vacunas Estreptocócicas/inmunología , Streptococcus pyogenes/inmunología , Vacunas Sintéticas/inmunología , Animales , Anticuerpos Antibacterianos/inmunología , Reacciones Cruzadas , Fagocitosis , ConejosRESUMEN
We have previously shown that a hexavalent group A streptococcal M protein-based vaccine evoked bactericidal antibodies after intramuscular injection. In the present study, we show that the hexavalent vaccine formulated with several different mucosal adjuvants and delivered intranasally induced serum and salivary antibodies that protected mice from intranasal challenge infections with virulent group A streptococci. The hexavalent vaccine was formulated with liposomes with or without monophosphorylated lipid A (MPL), cholera toxin B subunit with or without holotoxin, or proteosomes from Neisseria meningitidis outer membrane proteins complexed with lipopolysaccharide from Shigella flexneri. Intranasal immunization with the hexavalent vaccine mixed with these adjuvants resulted in significant levels of antibodies in serum 2 weeks after the final dose. Mean serum antibody titers were equivalent in all groups of mice except those that were immunized with hexavalent protein plus liposomes without MPL, which were significantly lower. Salivary antibodies were also detected in mice that received the vaccine formulated with the four strongest adjuvants. T-cell proliferative assays and cytokine assays using lymphocytes from cervical lymph nodes and spleens from mice immunized with the hexavalent vaccine formulated with proteosomes indicated the presence of hexavalent protein-specific T cells and a Th1-weighted mixed Th1-Th2 cytokine profile. Intranasal immunization with adjuvanted formulations of the hexavalent vaccine resulted in significant levels of protection (80 to 100%) following intranasal challenge infections with type 24 group A streptococci. Our results indicate that intranasal delivery of adjuvanted multivalent M protein vaccines induces protective antibody responses and may provide an alternative to parenteral vaccine formulations.